Ouabain-induced hypertension is characterized by hyperactivity of the sympathetic nervous system and increased contraction of vascular smooth muscle. It may also involve changes in sympathetic neuromuscular transmission in small arteries. The proposed research aims first to determine certain basic mechanisms of sympathetic transmitter release in small arteries, and then, to determine how these are affected by ouabain. Basic premises of the research are i) that sympathetic neuromuscular transmission and arterial contraction importantly involve the two co-transmitters, ATP and nor-epinephrine (NE) and, ii) that these two neurotransmitters are differentially released by Ca 2+ dependent mechanisms that are not yet completely known, possibly involving different synaptic vesicles, 'residual [Ca2+] ' and stored Ca 2+ (in addition to Ca 2+ entry). An overall hypothesis on mechanisms of ouabain actions is that inhibition of nerve terminal Na + pumps increases 'residual' [Ca 2+] and/or stored Ca 2+(thereby increasing NE and ATP release) and that the increases in terminal [Ca 2+] are mediated by Na/Ca exchange. Specific Aims are: 1) Determine the probabilities, at individual sympathetic nerve varicosities, of ATP and NE release, 2) Measure the sizes of ATP and NE transmitter packets ('quanta'), 3) Test the hypothesis that differential release of NE and ATP results from the release of different types of synaptic vesicles, 4) Test the hypothesis that acute, low-dose, ouabain inhibits the alpha3-isoform of the Na/K-ATPase in sympathetic varicosities and changes the probability of release, but not quantal size, 5) Determine whether release probability or quantal size is altered in ouabain hypertensive rats. Rat and mouse mesenteric small arteries will be loaded with fluorescent Ca 2+ indicators and studied in a myograph that permits simultaneous confocal fluorescence imaging, electrical stimulation/recording, and recording of isometric force development. Mice with genetically altered Na/Ca exchangers or ATP receptors will be used. Junctional Ca 2+ transients (jCaTs) will be used to measure neurally released ATP. Carbon fiber microelectrodes and amperometry will be used to measure NE release as 'NE oxidation currents' (NEOCs). The research will measure NE and ATP release together for the first time and thereby determine some of the basic mechanisms that control neurogenic contractions of arteries. will elucidate the mechanisms by which sympathetic nerves contribute to ouabain-induced hypertension.